Current standard treatments for bacterial infection rely predominantly on antibiotics. Under certain conditions, however, the use of antibiotics provoques the emergence or selection of resistant bacterial strains. Even worse, some bacterial strains are capable of developing resistance against entire panels of antibiotics. Therefore several programs have been launched to develop new companion compounds targeting bacterial virulence.
The mode of action of most antibiotics relies on the disruption of the bacterial growth cycle by preventing the synthesis or assembly of key components of bacterial processes such as cell wall synthesis, DNA replication and protein synthesis. Antibiotics are highly effective unless pathogens have become resistant against one or even multiple antibiotics. Today, multiresistant bacteria pose a major clinical problem and health threat (Health Care-Associated Infections, HAI). Infections due to antibiotic resistant microorganisms lead to significantly higher morbidity, longer hospitalization, increased mortality rates and increased health care costs. Especially the so called “ESKAPE” pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) represent a major harm to patients in hospitals. About 440,000 estimated HA infections among US adult inpatients annually result in additional healthcare cost of $9.8 billion dollar every year (Zimlichman et al. 2013).
Many nosocomial infections are caused by pseudomonas aeruginosa which is responsible for 10% of all hospital acquired infections (Aloush et al 2006). Infections caused by this microorganism are often life threatening and difficult to treat due to its low susceptibility to antimicrobial agents and to the frequent emergence of antibiotic resistance during therapy. These strains are sensitive to just a few antibiotic agents like cephalosporins, carbenicillin, colistin, gentamycin, polymyxin, quinolones and streptomycin (Sivanmaliappan et al 2011). Their prominent drug resistance results from de novo emergence of resistance after exposure to antimicrobials, and patient-to-patient contamination with resistant P. aeruginosa. Practically all known mechanisms of antimicrobial resistance can be observed, like: derepression of chromosomal AmpC cephalosporinase; production of plasmid or integrin mediated β lactamases from different molecular classes (carbenicillinases and extended spectrum β lactamases belonging to class A, class D oxacillinases and class B carbapenem hydrolysing enzymes); diminished outer membrane permeability (loss of OprD proteins); overexpression of active efflux systems with wide substrate profiles; synthesis of aminoglycoside modifying enzymes (phosphoryltransferases, acetyltransferases and adenylyltransferases); and structural alterations of topoisomerases II and IV determining quinolone resistance. Worryingly these mechanisms are often simultaneously developed and activated, thereby conferring multiresistant phenotypes, rendering this microbe less amenable to control in hospitals (Strateva et al 2009). According to data from the US Centers for Disease Control and Prevention and the National Nosocomial Infection Surveilance System, P. aeruginosa is the second most common cause of nosocomial pneumonia (17%), the third most common cause of urinary tract infections (7%), the fourth most common cause of surgical site infection (8%), the seventh most frequently isolated pathogen from the bloodstream (2%) and the fifth most common isolate (9%) overall from all sites (El Solh et al 2009). More importantly, it is the most common multidrug resistant Gram negative pathogen causing pneumonia in hospitalized patients.
To support the therapy of bacterial infections, bacterial virulence factors have become targets for reducing the symptoms of bacterial infections. Being essential for maintaining bacterial pathogenicity, virulence factors promote i.a. resistance to environmental threats and to host defense mechanisms, growth capability, adherence to the host, tissue specificity, and access to nutrition resources. A variety of bacterial and often strain specific components are involved, many of them harmful to the host. The coordinated function of virulence factors determines the aggressiveness of the strain. In many cases virulence factors are secreted proteins or enzymes, sometimes exhibiting very specific functions. For example, one of the most toxic bacterial virulence factors is the so called Lambda-toxin (light chain) secreted by Clostridium botulinum. The zinc-dependent protease is a thermolysin like protease (TLP) targeting synaptic vesicle fusion proteins and causing severe neurotoxic effects with a lethal dose as low eight nanograms per kilogram of body weight (Lebrun et al 2009). TLPs are present in many microorganism species, and many TLPs are regarded as key pathogenic factors involved in several severe bacterial infections. For example, Lambda-toxin from Clostridium perfringens degrades various human immune defense proteins. Vibriolysin from Vibrio spec. and Pseudolysin from Pseudomonas aeruginosa are potentially lethal blood-poisons in humans. Furthermore, Bacillus anthracis also disposes of potent metalloproteases.
Pseudosysin, aureolysin, bacillolysin, pseudolysin, vibriolysin, and anthrax npr599 belong to the M4 or metzincin family of metalloproteinases for which mammals and many invertebrates lack specific inhibitors. These proteases are presumably at the origin of many pathological symptoms associated with severe infections such as septicemia, hemorrhagic tissue bleeding, necrosis and enhancement of vascular permeability (Chung et al. 2006). Severe diseases like gastric and peptic ulcers and gastric carcinoma originate at least partly from the effect of pathogen M4 metalloproteinases (Schmidtchen et al. 2003, Smith et al. 1994).
Some virulence factors were recently recognized as putative targets for drug design and therapeutic intervention. While many newly discovered synthetic or natural small organic compounds exhibit anti virulence activity, antibodies neutralizing bacterial toxins are in the focus of current ant virulence strategies in industry. An example for this approach is the Humaneered® PEGylated, recombinant anti-Pseudomonas-PcrV antibody Fab′ fragment (KB001) that inhibits the function of the Pseudomonas aeruginosa type III secretion system (TTSS) (Milla et al. 2013). The PEGylation extends serum half-life and also protects against inactivation by proteases (mostly Pseudolysin) secreted by Pseudomonas aeruginosa at the target site. A clinical phase I/II trial for pneumonia prevention is currently ongoing. Another example is KBSA 301 from Kenta Biotech, a fully human IgG1 antibody highly specific for S. aureus exotoxin being active against MRSA and MSSA.
Inhibitors of pseudolysin were also identified and published. It was assumed that pseudolysin inhibitors could interfere with biofilm formation and preservation. Thus, all published in vitro tests were carried out as biofilm interference tests of pseudomonas a. N-mercaptoacetyl-Phe-Tyr-amide (K(i)=41 nM) (Cathgart G. R. et al, 2011), phosphoramidon, a compound produced by the Bacterium Streptomyces tanashiensis, and its derivative talopeptin, phosphonomadites, phenanthroline, and small molecules on a quinazolin basis (Khan et al 2009) all target pseudolysin, but also physiological M4 family related proteases of the host. Phosphoramidon, for example, is inhibiting the endogenous protein endothelin converting enzyme (ECE). These side effects are probably the main reason why none of these compounds was investigated further in clinical trials so far.
Cathgart et al. (2011) observed that a number of pseudolysin inhibitors can reduce already formed pseudomonas a. biofilms, whearas planktonic pseudomona a. was not at all affected by said inhibitors. They further observed that biofilms could be completely dissolved when said inhibitors were combined with antibiotics like gentamycin or ciprofloxacin, and both, inhibitor and antibiotic, were applied at high doses.
Popov et al. (Popov S. G. et al, 2005) published results of an experiment on the simultaneous application of an antibiotic compound (Ciprofloxacin, Bayer Healthcare AG) and metalloprotease inhibitors like phenanthroline and phosphoramidon in mice. They found that the two metalloproteinase inhibitors exerted an additional inhibition on the infectious strain, but only when applied with a delay of one or two days after the animal was challenged of with bacterial spores, and not immediately after the challenge. Immediate application of the inhibitor combination, however, resulted in just an insignificant difference in comparison to applying ciprofloxacin alone. Moreover the authors observed that applying pseudolysin inhibitors alone, i.e. without antibiotics in parallel, had no effect. These observations reflect the general expectation that the therapeutic effect of M4 protease inhibitors is limited to late infection stages when biofilms start forming and pseudolysin is shed. At these stages, a high dosis of both, inbitors and antibiotics, is expected to show strong inhibition of bacterial growth.
A particular peptide inhibiting thermolysin-like enzymes is the insect metalloproteinase inhibitor IMPIα. It was originally discovered in and purified from the hemolymph of immunized G. mellonella larvae (Wedde et al. 1998). Its active moiety comprises 69 amino acids including intramolecular cystein bonds, and a molecular weight of 7667.7 Da. The molecule has a reported IC60 of 0.62, 0.86 and only 81.66 nM for thermolysin, bacillolysin and pseudolysin, respectively. IMPIα was tested against human metallo-matrix proteases MMP1, 2, 3, 7, 8, and -9, of which only MMP1 and MMP2 showed a negligible inhibition. From this comparison it was further deduced that an active site loop would be present in IMPIα between aa 33-aa40, including a cleavage site between aa37 (Asparagine) and aa38 (Isoleucin). Other known protein inhibitors of metalloproteinases do not inhibit proteinases of the M4 protease family. It was shown that IMPIα can be produced recombinantly in bacteria, especially in E. coli. Eukaryotic cells such as CHO cells, transgenic plants and animals may also serve as sources for recombinant production. Alternatively, chemical synthesis of molecules and cell free recombinant production could also be means to produce IMPIα.
Vilcinskas A (2011) discloses in a review “Anti-Infective therapeutics from the lepidopteran model host Galleria mellonella” Current Pharmaceutical Design 17(13), 1240-124 that the larvae of the greater wax moth Galleria mellonella prosper in use both as surrogate alternative model hosts for human pathogens and as a whole-animal-high-throughput-system for in vivo testing of antibiotics or mutantlibraries of pathogens. In addition, a broad spectrum of antimicrobial peptides and proteins has been identified in this insect during the past decade among which some appear to be specific for Lepidoptera. Its arsenal of immunity-related effector molecules encompasses peptides and proteins exhibiting potent activity against bacteria, fungi or both, whose potential as new anti-infective therapeutics is presently being explored. Of particular interest is the insect metalloproteinase inhibitor (IMPI) which has been discovered in G. mellonella. The IMPI exhibits a specific and potent activity against thermolysin-like microbial metalloproteinases including a number of prominent virulence and/or pathogenic factors of human pathogens which are responsible for severe symptoms such as septicemia, hemorrhagic tissue bleeding, necrosis and enhancement of vascular permeability. The IMPI and antimicrobial peptides from G. mellonella may provide promising templates for the rational design of new drugs since evidence is available that the combination of antibiotics with inhibitors of pathogen-associated proteolytic enzymes yields synergistic therapeutic effects. The potential and limitations of insect-derived geneencoded antimicrobial compounds as antiinfective therapeutics are discussed.
Anja Clermont, et al discloses in Biochemical Journal August 2004, 382 (1) 315-322, in an article “Cloning and expression of an inhibitor of microbial metalloproteinases from insects contributing to innate immunity” that the first IMPI (inhibitor of metalloproteinases from insects) was identified in the greater wax moth, Galleria mellonella. They report cloning and expression of a cDNA coding for this IMPI. The IMPI mRNA was identified among the induced transcripts from a subtractive and suppressive PCR analysis after bacterial challenge of G. mellonella larvae. Induced expression of the IMPI during a humoral immune response was confirmed by realtime PCR, which documented up to 500 times higher amounts of IMPI mRNA in immunized larvae in comparison with untreated ones. The IMPI sequence shares no similarity with those of tissue inhibitors of metalloproteinases or other natural inhibitors of metalloproteinases, and the recombinant IMPI specifically inhibits thermolysin-like metalloproteinases, but not matrix metalloproteinases. These results support the hypothesis that the IMPI represents a novel type of immune-related protein which is induced and processed during the G. mellonella humoral immune response to inactivate pathogen-associated thermolysin-like metalloproteinases.
R. Caldwell et al. report in 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 1-10, 2003 about “Significant occurrences of arterial restenosis remain a complicating factor of endovascular stent implantation.” The incorporation of GM6001, a matrix metalloproteinase inhibitor (MMPI), into a poly(lactide-co-glycolide) (PLGA) absorbable coating for 316L stainless steel is proposed as a means to reduce the rate of smooth muscle cell proliferation and migration. Coatings were fabricated using a solvent evaporation technique, and the surfaces were characterized by noncontacting profilometry and scanning electron microscopy. Sufficient degradation of the PLGA determined by gel permeation chromatography occurred to release adequate amounts of the GM6001 from the coating within a 7-day period. A cumulative GM6001 release at day 42 was determined to be 77.8±1.4% of the actual GM6001 content within the coating. The coating containing the GM6001 reduced the rate of in vitro cell growth of human aortic smooth muscle cell (HASMC) by 30.7 and 37.4% compared to the metallic substrate only after 4 and 7 days, respectively. However, the MMP-2 activity normalized to cell number was not statistically different between the GM6001 releasing coating and the metal substrate. Using a scrape wound injury assay, the migration of HASMCs was shown to be decreased by 21.4% with GM6001 released from the PLGA coating compared to metallic substrate only. These results suggest that releasing a MMPI from an absorbable coating of a metallic substrate provides a reduction of HASMC proliferation and migration rates, while preserving the overall MMP activity in efforts to retain normal cellular regulation.
A. Vilcinskas et al. disclose in Entomological Research 37 (Suppl. I) (2007) A79 “The greater wax moth Galleria mellonella as a mini-host model for human pathogens and as a reservoir of novel peptide antibiotics.” The IMPI represents a specific inhibitor of microbial metalloproteinases that are virulence factors of human pathogens. It strongly inhibits prominent thermolysin-like metalloproteinases such aureolysin, bacillolysin, pseudolysin and vibriolysin which have been identified as targets for the development of second generation antibiotics (Clermont et al. 2004). Since thermolysin-like microbial metalloproteinases play well established roles during pathogenesis and cause pathologic Symptoms such as increase of vascular permeability, hemorrhagic edema, and septic injury, the IMPI represents a promising template for the design of novel peptide antibiotics (Wedde et al. 2007).
In summary, most known M4 protease inhibitors were found to exert activity against resistive bacteria only at later stages, whether alone or in combination with antibiotics.